strain: NDmed genotype: Wild-type, undomesticated condition: Adhesion time point: 3h
Treatment protocol
Wells were then rinsed with TSB to eliminate non-adherent bacteria and refilled with 200 µL of sterile TSB. For each time point (1h, 3h, 4h, 5h, 7h, 24h and 48h) 96-well plates were prepared.
Growth protocol
200 µL of an overnight culture of Bacillus subtilis NDmed in TSB (adjusted to an OD 600nm of 0.02) were added in each well of 96-well microtiter plates, incubated at 30°C for 90 min to allow the bacteria to adhere to the bottom of the wells.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted as described by Nicolas et al., (2012) Science 335, 1103–1106 (PMID:22383849). RNA concentration was measured using a NanoDrop spectrophotometer; RNA quality was checked by analysis with an Agilent 2100 Bioanalyzer (Agilent Technologies).
Label
Cy3
Label protocol
For cDNA synthesis, 10 µg of total RNA were mixed with random primers (FairPlay III Microarray Labeling Kit) and spike-ins (One-Color RNA Spike-In Kit, Agilent Technologies) and incubated at 70°C for 10 min followed by 5 min incubation on ice. Then, first-strand Master Mix, Actinomycin D (final conc. 40 µg/ml) and AffinityScript HC Reverse Transcriptase were added. The reaction was incubated for 60 min at room temperature and for 60 min at 42°C. After hydrolyzing the RNA, cDNA was precipitated overnight at -20°C. NHS-ester dye coupling (CyDye Mono-Reactive Dye, GE Healthcare) and purification of labeled cDNA were performed according to the FairPlay III instructions. cDNA and Cy-dye concentrations were quantified by means of a NanoDrop spectrophotometer.
Hybridization protocol
1200 ng of Cy3-labeled cDNA were hybridized to the tiling array following Agilent’s hybridization, washing and scanning protocol (One-Color Microarray-based Gene Expression Analysis, version 5.5).
Scan protocol
The microarray (BaSysBio Bacillus subtilis T3 array, 2x400K [Agilent-044473]) was scanned with Agilent Technologies Scanner, model G2505C. Grid: 044473_D_F_20121025. Protocol: GE1_107_Sep09.
Description
Sample of B. subtilis NDmed, 3 hour after adhesion.
Data processing
An aggregated expression value was computed for each GenBank annotated CDS and newly defined transcribed region as the median log2 expression signal intensity of probes lying entirely within the corresponding region as described in (Nicolas et al., 2012 PMID: 22383849). The expression intensity was computed from the raw intensity data using a model of signal shift and drift and correcting for probe affinity variations as described in (Nicolas et al., 2009, Bioinformatics 25, 2341-2347). Data were quantile normalized using LIMMA package. To control for possible cross-hybridization artefacts the sequence of each probe was BLAST-aligned against the whole chromosome sequence and probes with a SeqS value above the 1.5 cut-off were discarded (Wei et al., 2008 Nucl. Acids Res. 36, 2926-2938).
